The present invention concerns compositions and methods for the modulation of gene expression in plants, specifically using enzymatic nucleic acid molecules.
The following is a brief description of regulation of gene expression in plants. The discussion is not meant to be complete and is provided only for understanding of the invention that follows. This summary is not an admission that any of the work described below is prior art to the claimed invention.
There are a variety of strategies for modulating gene expression in plants. Traditionally, antisense RNA (reviewed in Bourque, 1995 Plant Sci 105, 125-149) and co-suppression (reviewed in Jorgensen, 1995 Science 268, 686-691) approaches have been used to modulate gene expression. Insertion mutagenesis of genes have also been used to silence gene expression. This approach, however, cannot be designed to specifically inactivate the gene of interest. Applicant believes that ribozyme technology offers an attractive new means to alter gene expression in plants.
Naturally occurring antisense RNA was first discovered in bacteria over a decade ago (Simons and Kleckner, 1983 Cell 34, 683-691). It is thought to be one way in which bacteria can regulate their gene expression (Green et al., 1986 Ann. Rev. Biochem. 55: 567-597; Simons 1988 Gene 72: 35-44). The first demonstration of antisense-mediated inhibition of gene expression was reported in mammalian cells (Izant and Weintraub 1984 Cell 36: 1007-1015). There are many examples in the literature for the use of antisense RNA to modulate gene expression in plants. Following are a few examples:
Shewmaker et al., U.S. Pat. Nos. 5,107,065 and 5,453,566 disclose methods for regulating gene expression in plants using antisense RNA.
It has been shown that an antisense gene expressed in plants can act as a dominant suppressor gene. Transgenic potato plants have been produced which express RNA antisense to potato or cassava granule bound starch synthase (GBSS). In both of these cases, transgenic plants have been constructed which have reduced or no GBSS activity or protein. These transgenic plants give rise to potatoes containing starch with dramatically reduced amylose levels (Visser et al. 1991, Mol. Gen. Genet. 225: 2889-296; Salehuzzaman et al. 1993, Plant Mol. Biol. 23: 947-962).
Kull et al., 1995, J. Genet. and Breed. 49, 69-76 reported inhibition of amylose biosynthesis in tubers from transgenic potato lines mediated by the expression of antisense sequences of the gene for granule-bound starch synthase (GBSS). The authors, however, indicated a failure to see any in vivo activity of ribozymes targeted against the GBSS RNA.
Antisense RNA constructs targeted against xcex94-9 desaturase enzyme in canola have been shown to increase the level of stearic acid (C18:0) from 2% to 40% (Knutzon et. al., 1992 Proc. Natl. Acad. Sci. 89, 2624). There was no decrease in total oil content or germination efficiency in one of the high stearate lines. Several recent reviews are available which illustrate the utility of plants with modified oil composition (Ohlrogge, J. B. 1994 Plant Physiol. 104, 821; Kinney, A. J. 1994 Curr. Opin. Cell Biol. 5, 144; Gibson et al. 1994 Plant Cell Envir. 17, 627).
Homologous transgene inactivation was first documented in plants as an unexpected result of inserting a transgene in the sense orientation and finding that both the gene and the transgene were down-regulated (Napoli et al., 1990 Plant Cell 2: 279-289). There appears to be at least two mechanisms for inactivation of homologous genetic sequences. One appears to be transcriptional inactivation via methylation, where duplicated DNA regions signal endogenous mechanisms for gene silencing. This approach of gene modulation involves either the introduction of multiple copies of transgenes or transformation of plants with transgenes with homology to the gene of interest (Ronchi et al. 1995 EMBO J. 14: 5318-5328). The other mechanism of co-suppression is post-transcriptional, where the combined levels of expression from both the gene and the transgene is thought to produce high levels of transcript which triggers threshold-induced degradation of both messages (van Bokland et al., 1994 Plant J. 6: 861-877). The exact molecular basis for co-suppression is unknown.
Unfortunately, both antisense and co-suppression technologies are subject to problems in heritability of the desired trait (Finnegan and McElroy 1994 Bio/Technology 12: 883-888). Currently, there is no easy way to specifically inactivate a gene of interest at the DNA level in plants (Pazkowski et al., 1988 EMBO J. 7: 4021-4026). Transposon mutagenesis is inefficient and not a stable event, while chemical mutagenesis is highly non-specific.
Applicant believes that ribozymes present an attractive alternative and because of their catalytic mechanism of action, have advantages over competing technologies. However, there have been difficulties in demonstrating the effectiveness of ribozymes in modulating gene expression in plant systems (Mazzolini et al., 1992 Plant Mol. Biol. 20: 715-731; Kull et al., 1995 J. Genet. and Breed. 49: 69-76). Although there are reports in the literature of ribozyme activity in plants cells, almost all of them involve down regulation of exogenously introduced genes, such as reporter genes in transient assays (Steinecke et al., 1992 EMBO J. 11:1525-1530; Perriman et al., 1993 Antisense Res. Dev. 3: 253-263; Perriman et al., 1995, Proc. Natl. Acad. Sci. USA, 92, 6165).
There are also several publications, [e.g., Lamb and Hay, 1990, J. Gen. Virol. 71, 2257-2264; Gerlach et al., International PCT Publication No. WO 91/13994; Xu et al., 1992, Science in China (Ser. B) 35, 1434-1443; Edington and Nelson, 1992, in Gene Regulation: Biology of antisense RNA and DNA, eds. R. P. Erickson and J. G. Izant, pp 209-221, Raven Press, NY.; Atkins et al., International PCT Publication No. WO 94/00012; Lenee et al., International PCT Publication Nos. WO 94/19476 and WO 9503404, Atkins et al., 1995, J. Gen. Virol. 76, 1781-1790; Gruber et al., 1994, J. Cell. Biochem. Suppl. 18A, 110 (X1-406) and Feyter et al., 1996, Mol. Gen. Genet. 250, 329-338], that propose using hammerhead ribozymes to modulate: virus replication, expression of viral genes and/or reporter genes. None of these publications report the use of ribozymes to modulate the expression of plant genes.
Mazzolini et al., 1992, Plant. Mol. Bio. 20, 715-731; Steinecke et al., 1992, EMBO J. 11, 1525-1530; Perriman et al., 1995, Proc. Natl. Acad. Sci. USA., 92, 6175-6179; Wegener et al., 1994, Mol. Gen. Genet. 245, 465-470; and Steinecke et al., 1994, Gene, 149, 47-54, describe the use of hammerhead ribozymes to inhibit expression of reporter genes in plant cells.
Bennett and Cullimore, 1992 Nucleic Acids Res. 20, 831-837 demonstrate hammerhead ribozyme-mediated in vitro cleavage of glna, glnb, glng and glnd RNA, coding for glutamine synthetase enzyme in Phaseolus vulgaris. 
Hitz et al., (WO 91/18985) describe a method for using the soybean xcex94-9 desaturasc enzyme to modify plant oil composition. The application describes the use of soybean xcex94-9 desaturase sequence to isolate xcex94-9 desaturase genes from other species.
The references cited above are distinct from the presently claimed invention since they do not disclose and/or contemplate the use of ribozymes in maize. Furthermore, Applicant believes that the references do not disclose and/or enable the use of ribozymes to down regulate genes in plant cells, let alone plants.
The invention features modulation of gene expression in plants specifically using enzymatic nucleic acid molecules. Preferably, the gene is an endogenous gene. The enzymatic nucleic acid molecule with RNA cleaving activity may be in the form of, but not limited to, a hammerhead, hairpin, hepatitis delta virus, group I intron, group II intron, RNaseP RNA, Neurospora VS RNA and the like. The enzymatic nucleic acid molecule with RNA cleaving activity may be encoded as a monomer or a multimer, preferably a multimer. The nucleic acids encoding for the enzymatic nucleic acid molecule with RNA cleaving activity may be operably linked to an open reading frame. Gene expression in any plant species may be modified by transformation of the plant with the nucleic acid encoding the enzymatic nucleic acid molecules with RNA cleaving activity. There are also numerous technologies for transforming a plant: such technologies include but are not limited to transformation with Agrobacterium, bombarding with DNA coated microprojectiles, whiskers, or electroporation. Any target gene may be modified with the nucleic acids encoding the enzymatic nucleic acid molecules with RNA cleaving activity. Two targets which are exemplified herein are delta 9 desaturase and granule bound starch synthase (GBSS).
Until the discovery of the inventions herein, nucleic acid-based reagents, such as enzymatic nucleic acids (ribozymes), had yet to be demonstrated to modulate and/or inhibit gene expression in plants such as monocot plants (e.g., corn). Ribozymes can be used to modulate a specific trait of a plant cell, for example, by modulating the activity of an enzyme involved in a biochemical pathway. It may be desirable, in some instances, to decrease the level of expression of a particular gene, rather than shutting down expression completely: ribozymes can be used to achieve this. Enzymatic nucleic acid-based techniques were developed herein to allow directed modulation of gene expression to generate plant cells, plant tissues or plants with altered phenotype.
Ribozymes (i.e., enzymatic nucleic acids) are nucleic acid molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence-specific manner. Such enzymatic RNA molecules can be targeted to virtually any RNA transcript, and efficient cleavage has been achieved in vitro and in vivo (Zaug et al., 1986, Nature 324, 429; Kim et al., 1987, Proc. Natl. Acad. Sci. USA 84, 8788; Dreyfus, 1988, Einstein Quarterly J. Bio. Med., 6, 92; Haseloff and Gerlach, 1988, Nature 334 585; Cech, 1988, JAMA 260, 3030; Murphy and Cech, 1989, Proc. Natl. Acad. Sci. USA., 86, 9218; Jefferies et al., 1989, Nucleic Acids Research 17, 1371).
Because of their sequence-specificity, trans-cleaving ribozymes may be used as efficient tools to modulate gene expression in a variety of organisms including plants, animals and humans (Bennett et al., supra; Edington et al., supra; Usman and McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Ribozymes can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the mRNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a particular phenotype and/or disease state can be selectively inhibited.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.